Anaplastic thyroid cancer (ATC) remains one of the solid tumors that is associated with the poorest prognosis. Standard therapy includes maximal safe resection, external beam radiation therapy (EBRT), and cytotoxic chemotherapy. Despite this, there are high rates of disease recurrence. Local/regional recurrence is particularly difficult for patients with ATC, resulting in airway and/or esophageal compromise which contributes to mortality and metastatic dissemination. Novel therapies are thus needed to improve disease control and lengthen survival. Recently, genomic profiling of ATC has uncovered high frequency mutations in the RAS-RAF-MEK-ERK pathway (particularly BRAF and RAS), as well as other DNA damage and cell cycle checkpoint control genes, including TP53. Our preclinical data supports that an activating BRAFV600E mutation promotes resistance to EBRT and genotoxic therapies, through the non-homologous end-joining repair (NHEJ) DNA repair pathway. In addition, targeted inhibition of BRAFV600E with a small molecule inhibitor results in sensitization to EBRT in BRAF mutant (BRAFm) ATC. Furthermore, treatment of BRAF mutant cells with a MEK-1/2 inhibitor also results in radiosensitization. Since BRAF wild-type (BRAFwt) ATC accounts for ~ 60-70% of cases, developing targeted strategies for radiosensitization in BRAFwt ATC is also critical. As such, we find that TP53 mutant ATC is effectively radiosensitized by ATR and Wee1 kinase inhibitors, highlighting the dependency of these tumors on the G2/M cell cycle checkpoint. Finally, we will explore radiation sensitization approaches for RAS mutant ATC, another common BRAFwt molecular subtype of ATC. In this proposal, we will attempt multiple strategies to advance therapy for patients with BRAFm and BRAFwt ATC. In Aim 1, we will perform a phase I trial to determine the maximally-tolerated doses of dabrafenib (BRAF inhibitor) and trametinib (MEK-1/2 inhibitor) to be used concurrently with EBRT for BRAFm ATC, and identify biomarkers of response and molecular pathways leading to resistance. In Aim 2, we will perform mechanistic studies to better understand how BRAFm leads to accelerated DNA repair, test novel therapeutic strategies targeting components of DNA repair, and develop and optimize novel strategies targeting DNA repair in combination with EBRT and other genotoxic therapies for BRAFm ATC. In Aim 3, we will attempt to develop novel strategies for treating BRAF wild-type ATC, by testing different targeted strategies for TP53 and RAS deficient or mutated ATC in vitro and in vivo to support future clinical testing of these combinations. Together, these studies will improve our understanding of how BRAF mutations impart radio-resistance, and identify new tumor-selective combinatorial approaches for treating patients with BRAF mutant and BRAF wild-type ATC.